Effects of host-associated low-temperature probiotics in olive flounder (Paralichthys olivaceus) aquaculture

This study investigated the effects of supplementation of low-temperature probiotics isolated from the intestines of olive flounder on the growth performance, digestibility, and regulation of intestinal microbiota and the expression of genes related to growth, immunity, and apoptosis in olive flounder. Bacteria showing high growth at approximately 15–20 °C, which is the temperature of olive flounder culture, were isolated and confirmed to be Pseudomonas species through 16S rRNA gene sequence analysis. Whole-genome sequencing revealed that the strain has a 6,195,122 bp single circular chromosome and a guanine–cytosine content of 59.9%. In the feeding trial, supplementation with 1 × 108 CFU/g of the isolate strain positively modulated growth performances, digestive enzyme activity, and gut microbiota composition of olive flounder. RT-qPCR for the comparison of growth, immunity, and apoptosis-related gene expression levels showed no significant differences between the groups. Therefore, the isolated host-associated low-temperature probiotics improved the growth performance of olive flounder by causing positive changes in digestive activity and intestinal microbial composition without affecting host gene expression.


Bacterial isolation and identification
Bacteria were isolated from the intestine of wild olive flounder caught off the coast of Yeongdo (35°04′16.2″N 129°05′08.0″E).Briefly, the intestine was homogenized and suspended in phosphate-buffered saline (PBS).The suspension was serially diluted with PBS, poured into a brain heart infusion (BHI) agar plate, which is rich in nutrients and can cultivate various microorganisms, and incubated at 20 °C for 24 h.The cultured single colony was identified by performing 16S ribosomal RNA gene sequence analysis, mixed with 50% glycerol, and stored at − 80 °C until it was used.

Bacterial whole genome sequencing
The whole-genome sequence (WGS) of the isolated bacteria was analyzed.The total DNA of the selected bacteria was extracted using the NucleoSpin Microbial DNA Mini kit (Macherey Nagel, Germany) 21 and sequenced using an Oxford Nanopore MinION (NFEC-2022-08-281084) at the KNU NGS Center (Daegu, South Korea).

Experimental diet preparation
The basal diet composition is shown in Table 1.The raw materials and composition ratios of the feed used were provided by the National Institute of Fisheries Science (Pohang, South Korea).FM70 with 70% fishmeal content and FM35 with 35% fishmeal content were prepared.A HALP-added feed was made by spraying on FM35, and the added probiotic concentration was 1 × 10 8 CFU/g.It was sprayed and dried before feeding every day.

Animals and experimental management
A total of 270 juvenile olive flounders with an average weight of 10.83 ± 0.26 g were obtained from a commercial hatchery (Jeogu Susan, South Korea).After 2 weeks of acclimatization, fish were randomly divided into 120 L semi-recirculating tanks (30 fish/tank, triplicates) and fed with the feed twice daily up to apparent satiation.The following aquatic environmental parameters were maintained throughout the experiment: temperature, 20.0 °C ± 0.5 °C; pH, 7.3 ± 0.3; salinity, 31 ± 1 ppt; and dissolved oxygen, 6.0 ± 0.5 mg/L.Seawater was provided after pre-filtration and UV sterilization.

Growth performance, feed utilization, and body indices
Growth performance, feed utilization, and body indices were calculated by measuring fish weight, length, visceral weight, and liver weight after 24 h of fasting on the last day of week 8.These parameters were calculated as follows: Weight gain (WG, %) = final weight − initial weight /initial weight × 100

Serum nonspecific immune and biochemical parameter analysis
Superoxide dismutase (SOD) activity was expressed as the percentage of superoxide inhibition by using a SOD activity colorimetric assay kit (BioVision, USA) in accordance with the manufacturer's instructions.The respiratory burst (RB) generated by phagocytes was measured via a nitro blue tetrazolium assay (Sigma-Aldrich, USA) as previously described 22 .Myeloperoxidase (MPO) activity was analyzed with an MPO colorimetric activity assay kit (Sigma-Aldrich, USA).Antiprotease activity was expressed as the percentage of trypsin inhibition by using the method of Ellis 23 .Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total glucose, total protein, triglyceride, and total cholesterols levels were measured with a BS-390 chemistry analyzer (Mindray Bio-Medical Electronics, China) at the Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, South Korea).

Digestive enzyme activity
Intestinal digestive enzyme activity was analyzed in fish midgut samples and evaluated using amylase, lipase, and protease assay kits (BioVision, USA) in accordance with the manufacturer's instructions.

Intestinal microbiota analysis
Total bacterial DNA was extracted from the intestines of olive flounder randomly selected from each group by using the PureHelix™ genomic DNA prep kit (NanoHelix, South Korea).The quality of the extracted DNA was examined using a NanoDrop Lite spectrophotometer (Thermo Scientific, USA) and gel electrophoresis.A library was constructed in accordance with 16S Metagenomic Sequencing Library Preparation (Illumina, USA), and the sequence of the V3-V4 region was analyzed using the Illumina MiSeq System (Illumina, USA).Sequence data were analyzed with the Quantitative Insights into Microbial Ecology pipeline (http:// qiime.org) via the SILVA database.The divisive amplicon denoising algorithm 2 pipeline was used to filter, denoise, merge, and remove chimeras of sequences.All samples generated rarefaction curves to confirm the tendency toward saturation, and sufficient sequence depth was set.

Gene expression analysis
Gene expression in the brain, kidney, and intestine was analyzed using RT-qPCR (Thermal cycler dice real time system, Model TP700/760, Takara, Japan).RNA was isolated from each organ by using a Hybrid-R™ RNA purification kit (GeneAll Biotechnology, South Korea).After the remaining DNA was removed with a Riboclear™ plus kit (GeneAll Biotechnology, South Korea), purity and concentration were evaluated with a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific).cDNA was synthesized with a PrimeScript™ first-strand cDNA synthesis kit (Takara, Japan), and RT-qPCR was performed using primers based on the gene sequence in Table 2 and TB Green™ Premix Ex Taq (Takara, Japan).Relative quantification was calculated by the 2 − ΔΔCT method using β-actin as a reference gene.

Statistical analysis
Data were statistically analyzed via ANOVA by using Statistical Package for the Social Sciences (SPSS; IBM, USA), followed by Duncan's multiple range test.Results with P < 0.05 were considered significant.www.nature.com/scientificreports/

Ethics approval and consent to participate
This study was conducted under the guidelines of the Animal Ethics Committee Regulation issued by Dong-Eui University (DEU-R2022-031).

Bacterial isolation, identification, and characteristics
In bacterial identification based on 16S rRNA sequences, the isolated bacteria shared 100% homology with Pseudomonas azotoformans DSM 18862 T (MNPV01000020), P. carnis B4-1 T (MNPV01000020), and P. paralactis DSM 29164 T (KP756921; Fig. 1A).The isolated strain was named Pseudomonas sp.BA28.In general, BA28 can survive at 10 °C-40 °C and pH 4-8.Its optimal growth conditions were identified as 15 °C and pH 6.Its complete genome was circular and had 6,195,122 bp with a 59.9% guanine-cytosine (GC) content.It also had 6,752 protein-encoding genes and 88 RNAs. Figure 1B shows a circular plot of the genome, including the number of bases, GC skew, GC content, and location of all annotated open reading frames sorted by the clusters of orthologous gene (COG) category and colored.

Growth performance, feed utilization, and body indices
The growth performance and feed utilization parameters of the FM70 and FM35 groups had no significant differences.Conversely, the HALP group supplemented with probiotics showed positive changes compared with those of the two other groups.The final body weight of the HALP group was 37.59 ± 1.29 g, which was significantly increased compared with those of the FM70 (32.21 ± 0.46 g) and FM35 (31.24 ± 0.82 g) groups.
Variations in the final body weight caused significant differences in the weight gain and specific growth rate among the three groups.The feed conversion ratio of the HALP group (1.06 ± 0.07) was significantly decreased compared with those of the FM70 (1.25 ± 0.03) and FM35 (1.26 ± 0.06) groups.The protein efficiency ratio of the HALP group (1.85 ± 0.13) was significantly increased compared with those of the FM70 (1.57± 0.04) and FM35 (1.55 ± 0.10) groups.The condition factor and organosomatic indices did not significantly differ among the three groups (Table 3).

Serum nonspecific immune and biochemical parameter analysis
The nonspecific immune parameters in the respiratory burst showed significant differences.Such parameters in the HALP group (0.66 ± 0.05) increased compared with those in the FM70 (0.56 ± 0.04) and FM35 (0.52 ± 0.03) groups.Other parameters (superoxide dismutase, myeloperoxidase, and antiprotease activity; Fig. 2) did not significantly differ among the three groups.All biochemical parameters did not also significantly vary among the three groups (Fig. 3).

Digestive enzyme activity
Digestive enzyme activities, specifically amylase and protease activities, in the intestine showed significant differences.The amylase activity significantly increased in the HALP group (8.08 ± 0.84 U/g) compared with that in the FM70 (3.49 ± 0.45 U/g) and FM35 (4.11 ± 0.94 U/g) groups.The protease activity significantly differed only between the HALP (4.64 ± 0.88 U/g) and FM70 (2.61 ± 1.08 U/g) groups.The lipase activity did not significantly differ among the three groups (Fig. 4).www.nature.com/scientificreports/

Intestinal microbiota analysis
All α-diversity estimates (ACE, Chao1, Jackknife, Shannon, and Simpson) did not significantly differ among the three groups (Fig. 5A,B).β-diversity analysis based on the UniFrac metric via principal coordinate analysis revealed clear boundaries between the three groups.The FM70 group was separated from the two other groups in the first PC, and the separation from the FM35 and HALP groups was confirmed in the second PC (Fig. 5C).The difference between the three groups in the composition of bacteria at the phylum level is the relative abundance of Proteobacteria and Firmicutes.Proteobacteria and Firmicutes accounted for 92.41% and 6.06% in the FM70 group, 64.44% and 24.72% in the FM35 group, and 54.50% and 44.05% in the HALP group, respectively (Fig. 5D).At the genus level, 73.90% of the total bacterial communities in the FM70 group were Comamonadaceae_uc and 22.72% and 24.52% in the FM35 and HALP groups, respectively.The relative abundance of Lactococcus and Lactobacillus was higher in the FM35 (20.24% and 3.80%) and HALP (37.14% and 6.16%) groups than in the FM70 (2.59% and 3.06%) group (Fig. 5E).

Gene expression analysis
RT-qPCR was performed to compare the expression levels of growth-and immune-related genes in olive flounder fed with each experimental diet.The gene expression of growth hormone in the brain and the growth hormone receptor in the kidney and intestine did not significantly differ in all groups (Fig. 6A).Immune-related gene expression levels between the three groups were compared in the kidney and intestine.The expression levels of the five genes did not significantly vary among the three groups (Fig. 6B,C).The expression levels of the apoptosis-related genes in the kidney and intestine did not significantly differ among the three groups (Fig. 7).

Discussion
In this study, HAPs were isolated from the intestine of healthy wild olive flounder, and the isolated strain was identified as a Pseudomonas species through whole genome sequencing.Various probiotic strains for farmed fish have been studied, but studies on HAPs are rare.They can adapt to the intestinal environment of the host better than candidate strains from other sources and cause various effects, such as increasing the rate of nutrient     digestion and absorption by producing enzymes and metabolites that show high activity in the host's intestinal environment 17,21,24 .In the present study, low-temperature probiotics showing the optimal growth rate at 15-20 °C were selected as candidate strains.Most probiotics, including Bacillus species and lactic acid bacteria, developed for farmed olive flounder are strains that show high growth rates at approximately 37 °C3,10,11,19,20 .However, evidence is insufficient to show whether these strains as probiotics provide beneficial effects to the host at 15-20 °C, which is the growth temperature of olive flounder.In this context, the host-associated low-temperature probiotics proposed in this study for the first time would be suitable for the development of probiotics for farmed fish in terms of their ability to colonize in the host's intestine and provide beneficial effects.Probiotics studied in farmed fish provide many positive effects, including improved growth performance 19 .Probiotics not only improve the appetite of farmed fish but also increase their digestibility by breaking down indigestible substances such as phytic acid 25 .Consequently, their growth increases, and feed utilization improves.Liu et al. 26 demonstrated that Bacillus species produce various enzymes that efficiently metabolize carbohydrates, lipids, and proteins, and their activities help improve the growth performance of the host.In the present study, BA28 was confirmed to have various enzyme genes related to carbohydrate, protein, and lipid metabolism through WGS analysis.In addition, microorganisms such as Pseudomonas species that survive at relatively low temperatures are known to produce enzymes that show higher activity at low temperatures than those produced by Bacillus species, which have an optimum temperature of 37 °C.In fact, phytase produced by Bacillus species shows a high activity at 50 °C or higher, whereas phytase produced by Pseudomonas species exhibits a high activity at 25-40 °C27 .This finding indicates that enzymes produced by Pseudomonas species that survive at low temperatures are more appropriate than those produced by Bacillus species to increase digestibility by decomposing phytic acid, an indigestible substance in the intestine of olive flounder living at approximately 20 °C27 .These results suggest that probiotics with high growth at a temperature similar to the growth temperature of the host have more potential to provide beneficial effects; in the case of olive flounder, low-temperature probiotics with high growth at 15-20 °C are more effective than high-temperature probiotics.
Among the many beneficial effects of probiotics, immune system modulation is one of the most recognized benefits 28 .Immunological studies on several probiotic-fed fish have shown that several probiotics, individually or in combination, can improve fish immunity 28 .Conversely, the provision of probiotics in this study did not cause significant differences in SOD, MPO, and antiprotease activity among the innate immunity parameters of flounder.The probiotics used in this study were not newly introduced strains in the intestines of flounder, but they are existing microorganisms isolated from the intestines of flounder; therefore, they may not affect the regulation of innate immunity.However, few studies have been conducted on the effects of HAP in fish.Therefore, more research is needed to fully understand the effects of HAPs on the regulation of innate immunity in fish.
Probiotics produce various enzymes that aid the host's digestion; when applied appropriately to fish, they not only increase feed efficiency and growth but also eliminate antinutritional factors present in the feed and prevent intestinal disorders and pre-digestion 29 .Sankar et al. 30 reported that microbial-derived exogenous enzymes have a wide pH range, are active throughout digestion, and may allow for better substrate hydrolysis.Thus, they suggest supplementing the diet with probiotics may improve digestive enzyme activities 30 .As described above, the probiotics used in this study were derived from the host's intestine, suggesting that they adapt well to the host's intestinal environment.Moreover, the enzymes they produce likely remain highly active in the host's intestinal environment.As a result, these properties can increase the digestibility of olive flounder by increasing digestive enzyme activities.www.nature.com/scientificreports/ The gut microbiome influences the host's gut development, immunity, and metabolism, and an imbalanced fish gut microbiome causes decreased metabolism, growth, stress, and disease development 31 .The gut microbiome is primarily formed by diet and can be modified by antibiotics, immunostimulants, prebiotics, and probiotics 32,33 .Among the changes induced by HALP supplementation in this study, a notable part was the increase in the relative abundance of lactic acid bacteria such as Lactobacillus and Lactococcus.Lactic acid bacteria are generally recognized as beneficial microorganisms because of their ability to stimulate the host's gastrointestinal tract development, digestive function, mucosal tolerance, and disease resistance 34 .Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Streptococcus, Carnobacterium, Weissella, and Pediococcus exist as indigenous species in the intestine of fish, and they have attracted attention as probiotics for aquaculture because they can produce antibacterial substances against potential fish pathogens.Therefore, the HALP dietary supplementation can provide positive effects by increasing the beneficial bacteria in the intestine of flounder, and the increase in growth performance and digestive enzyme activities may be the result of changes in the intestinal microbiota.Although Pseudomonas species used as HALPs in this study were confirmed to increase the abundance of LAB by causing changes in the composition of the intestinal microflora of flounder, the exact mechanism could not be determined from the results of this study.Previous studies have reported that bacteriocins, such as pediocins, produced by LAB can exert antagonistic effects on a variety of bacteria, including gram-negative and gram-positive species, thereby increasing their relative abundance [35][36][37] .Nevertheless, because the strain used in this study is not a LAB, further research is needed to determine the mechanism of these changes.
Growth hormone and growth hormone receptor play an important role in fish growth and can be used as good biomarkers for estimating growth rates in various fish 38,39 .Previous studies have reported that probiotic supplementation can affect growth and immune-related gene expression in fish 40,41 .In the present study, the comparison of growth-related gene expression revealed no significant difference in any group, confirming no genetically related differences in growth and proving that the increase in growth by HALPs was not associated with the gene expression level.These results indicate that the increase in the growth performance of fish in this study was independent of gene expression and may be related to nutrient digestion and absorption.This could have the advantage of being safe to use, as there are no other physiological effects that may occur due to changes in gene expression.Similarly, a study using a cell-based laboratory model of the salmon gut reported that beneficial microbes had no effect on the expression of key intestinal barrier and immune molecules 42 .
Various probiotics affect the expression of immune-related genes in fish 19 .Previous reports showed that the moderate transcription of immune-related genes, such as pro-inflammatory cytokines, helps maintain immune balance and increase resistance to infection 19,43 .Peptidoglycan, lipopolysaccharide, flagella, and nucleic acids, which are constituents of various probiotics, are known as microbe-associated molecular patterns (MAMPs) that induce the transcription of pro-inflammatory cytokines 19,44 .The binding of MAMPs to pathogen pattern recognition receptors on dendritic cells or Toll-like receptors activates and stimulates macrophages and T cells to induce the transcription of pro-inflammatory cytokines [45][46][47] .However, similar to the innate immune response in this study, immune-related gene expression was not significantly affected by the dietary supplementation of HALPs.Since HALPs were isolated from the gut of the host, they did not appear to stimulate the host's immunerelated response.Similarly, HALPs did not significantly affect apoptosis-related gene expression, indicating that they did not exert any toxic effect on host cells.

Conclusion
The HALPs isolated in this study improved the growth performance of olive flounder by positively changing the digestive activity and intestinal microbial composition without affecting host gene expression.They could be used as novel feed additives for olive flounder farming.

Figure 1 .
Figure 1.Phylogenetic tree analysis and circular genome visualization of Pseudomonas sp.BA28.(A) The phylogenetic tree based on 16S rRNA gene sequences was constructed by the neighbor joining method using MEGA 7 with 1000 bootstrap repetitions, after which the 16S rRNA sequences were aligned by the Clustal W program. (B) Circles from the center to the outside: the number of bases, GC skew, GC content, location of all annotated ORFs (colored by COG categories).

Table 3 .
Growth performance, feed utilization and organosomatic indices of olive flounder fed the experimental diets.WG weight gain = 100 × (Final weight − Initial weight)/Initial weight, SGR specific growth rate = 100 × (ln final weight − ln initial weight)/days, FCR feed conversion ratio = Dry feed intake/Wet body WG, PER protein efficiency ratio = Wet body WG/Protein fed, CF condition factor = 100 × Body weight/ (Total body length)3, VSI viscerosomatic index = 100 × (Visceral weight/Body weight), HSI hepatosomatic index = 100 × (Liver weight/Body weight), Values are mean ± SD of three replicates.Values with different superscript letters within the same column in the table are significantly different (P < 0.05).The lack of superscript letter indicates no significant differences (P > 0.05).FM70, basal diet; FM35, low-fishmeal diet; HALP, low-fishmeal diet supplemented with host-associated low-temperature probiotics.

Figure 4 .
Figure 4. Amylase (A), lipase (B), and protease (C) activities in intestine after 8-month feeding trial in olive flounder.Data represent the mean ± standard deviation; means that do not share the same letter differ significantly (P < 0.05).FM70 basal diet, FM35 low-fishmeal diet, HALP low-fishmeal diet supplemented with host-associated low-temperature probiotics.

Figure 5 .
Figure 5. Intestinal microbiota analysis of olive flounder fed experimental diet.α-diversity of the bacterial communities (A,B).β-diversity based on principal coordinates analysis (C).Composition and relative abundance of intestinal bacterial communities at the phylum (D) and genus (E) level.ns means no significant difference (P > 0.05).FM70 basal diet, FM35 low-fishmeal diet, HALP low-fishmeal diet supplemented with host-associated low-temperature probiotics.

Figure 6 .
Figure 6.Profiles of growth (A) and immune (B,C) related genes expression of olive flounder as measured by RT-qPCR.The gene expression level of growth hormone (GH) was investigated in the brain, and the gene expression of growth hormone receptor (GHR) was investigated in the kidney (GHT-K) and intestine (GHT-I).Immune-related gene expression was investigated in both kidney (B) and intestine (C).Gene expression was quantified relative to β-actin transcription.Data represent the mean ± standard deviation of 3 replicates (3 fish/replicate); ns means no significant difference (P > 0.05) and star means significant difference (P < 0.05).FM70 basal diet, FM35 low-fishmeal diet, HALP low-fishmeal diet supplemented with host-associated lowtemperature probiotics, IL interleukin, TNF tumor necrosis factor.

Figure 7 .
Figure 7. Profiles of apoptosis related genes expression of olive flounder as measured by RT-qPCR.The gene expression level was investigated in the kidney (A) and intestine (B).Gene expression was quantified relative to β-actin transcription.Data represent the mean ± standard deviation of 3 replicates (3 fish/replicate); ns means no significant difference (P > 0.05).FM70 basal diet, FM35 low-fishmeal diet, HALP low-fishmeal diet supplemented with host-associated low-temperature probiotics, Cas caspase, FasL FAS ligand.

Table 1 .
Dietary formulation of the experimental diets for juvenile olive flounder.

Table 2 .
Gene specific primers used to quantify relative gene expression.